Process of making transgenic mammals that produce exogenous proteins in milk and transgenic mammals produced thereby

Abstract

The invention relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

1 . A plasmid comprising a gene encoding a protein of interest operably linked to a beta casein promoter and a β lactamase gene. 
     
     
         2 . The plasmid of  claim 1 , wherein said protein of interest is a growth hormone. 
     
     
         3 . The plasmid of  claim 2 , wherein said growth hormone is a mammalian growth hormone. 
     
     
         4 . The plasmid of  claim 3 , wherein said mammalian growth hormone is human growth hormone, bovine growth hormone, porcine growth hormone, ovine growth hormone, caprine growth hormone or rodent growth hormone. 
     
     
         5 . The plasmid of  claim 4 , wherein said mammalian growth hormone is human growth hormone. 
     
     
         6 . The plasmid of  claim 5 , which is pRβhGH. 
     
     
         7 . The plasmid of  claim 6 , further comprising a neomycin resistance gene. 
     
     
         8 . The plasmid of  claim 7 , which is pRNeo. 
     
     
         9 . The plasmid of  claim 8 , further comprising a gene encoding a green fluorescent protein (GFP) operably linked to a cytomegalovirus (CMV) promoter. 
     
     
         10 . The plasmid of  claim 9 , which is pRNeoGreen. 
     
     
         11 . The plasmid of  claim 5 , wherein the beta casein promoter comprises 1230 bp of the full-length beta casein promoter and 49 bp of the first non-coding exon of the beta casein gene. 
     
     
         12 . The plasmid of  claim 11 , further comprising a neomycin resistance gene. 
     
     
         13 . The plasmid of  claim 12 , which is pVEβcashGH. 
     
     
         14 . A linear fragment of the plasmid according to  claim 8 , wherein said β lactamase gene was excised by digestion with ApaLI. 
     
     
         15 . A linear fragment of the plasmid according to  claim 10 , wherein said β lactamase gene was excised by digestion with ApaLI. 
     
     
         16 . A linear fragment of the plasmid according to  claim 13 , wherein said β lactamase gene was excised by digestion with ApaLI. 
     
     
         17 . A plasmid selected from the group consisting of pRβphGH, prNeo, and pRNeo Green. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A method for transfection of genetic constructs according to  claim 1  into mammalian cells, comprising the combination of cationic lipids for liposome utilization. 
     
     
         21 - 61 . (canceled) 
     
     
         62 . A method of making a non-human transgenic mammal comprising:
 a) superovulating a female non-human mammal which is transgenic for the production of a recombinant growth hormone in its milk;   b) artificially inseminating said mammal with semen obtained from a male non-human, non-transgenic mammal, to produce embryos;   c) collecting said embryos;   d) implanting said embryos in the uterus of a receptive mammal; and   e) monitoring the pregnancy through the birth of the transgenic mammal.   
     
     
         63 . A method of making a non-human transgenic mammal comprising:
 a) superovulating a female non-human mammal which is transgenic for the production of a recombinant growth hormone in its milk;   b) artificially inseminating said mammal with semen obtained from a male non-human mammal which is transgenic for the production of said recombinant growth hormone, to produce embryos;   c) collecting said embryos;   d) implanting said embryos in the uterus of a receptive mammal; and   e) monitoring the pregnancy through the birth of the transgenic mammal.   
     
     
         64 . A method of making a non-human transgenic mammal comprising:
 a) superovulating a female non-human, non-transgenic mammal;   b) artificially inseminating said mammal with semen obtained from a male non-human mammal which is transgenic for the production of a recombinant growth hormone, to produce embryos;   c) collecting said embryos;   d) implanting said embryos in the uterus of a receptive mammal; and   e) monitoring the pregnancy through the birth of the transgenic mammal.   
     
     
         65 - 77 . (canceled)